Chrysanthemum is one of the important cut flower worldwide. It ranks 3rd in world among the cut flowers. Chrysanthemum is commonly propagated vegetatively and this practice allows the viruses, once established in the plants to be perpetuated from generation to generation. Quality of germplasm and minimizing the infection of the viruses to different cultivars, proper diagnosis and control for viral diseases are not only desirable but also essential for improving crop productivity.
Chrysanthemum virus B (CVB), a carlavirus has a narrow host range and distributed worldwide wherever Chrysanthemums are grown. It infects Chrysanthemum and about 10 other species in 5 dicotyledonous families (Brunt, A. A., Crabtree, K., Dallwitz, M. J., Gibbs, A. J., and Watson, L. (Edts) Viruses of Plants, CAB International, UK. Page No. 398-400). CVB is widespread throughout the country. During a survey of chrysanthemum cvs. all tested commercial stocks were found to show disease incidence ranging between 40% to 95% (Verma, N., Sharma, A., Ram, R., Hallan, V., Zaidi, A. A. and Garg, I. D. (2003) Detection, identification and incidence of Chrysanthemum B carlavirus in chrysanthemums in India. Crop Prot. 22, 425-429). At the Institute of Himalayan Bioresource Technology, Palampur chrysanthemum cultivars were collected from twenty eight geographical areas. Twenty eight isolates were cloned, sequenced and sequences were submitted to Genbank. Different primer pairs were designed (EMBL Nucleotide Sequence Accession Numbers: AJ566196, AJ566195, AJ609493, AJ609494, AJ609495, AJ609496, AJ609497, AJ609498, AJ609499, AJ609500) and used successfully for identification and characterization of an Indian isolates of CVB. This also shows wide spread occurrence of CVB in chrysanthemum being cultivated in different parts of country.
CVB was first reported from Netherlands as a member of carlavirus group (Noordam, D. 1952 Virusziekten bij chrysant in Nedeland. With a summary: virus disease of Chrysanthemum morifolium in Netherlands. Tijdschrift over Plantenziekten. 58, 121-190). CVB infection results in loss of flower quality, mild leaf mottling, vein clearing or a combination of these is found (Hollings, M. and Stone, O. M. (1972) Chrysanthemum virus B CMI/AAB Descriptions of Plant viruses No. 110). Symptoms ranging from mosaic, malformation and slight to severe necrosis have also been reported (Hakkart, F. A. and Matt, D. Z. (1974) Variation of Chrysanthemum virus B. J. Plant Path. 80, 97-103).
Traditional methods of diagnosis of plant viruses require bioassay through an indicator plant, symptom observation, host range determination, and particle morphology and vector relations. These processes are time consuming and require a lot of labour. However, progress in molecular biology, biochemistry and immunology has led to the development of new accurate, rapid and less labour-intensive methods of virus detection. There are various diagnostic techniques available in the field of viral diagnostics like precipitation tests, agglutination tests, fluorescent antibody test, enzyme linked immunosorbant assay, dot immunosorbant assay, tissue blotting assay, western blotting, nucleic acid hybridization with radio labeled and non radio-labeled probes and polymerase chain reaction based detection.
Immunological techniques have been successfully used for the detection of CVB from Chrysanthemum morifolium (Raizada, R. K., Srivastava, K. M., Chandra, G. and Singh, B. P. (1989) Comparative evaluation of sero-diagnostic methods for detection of Chrysanthemum virus B in chrysanthemum. Indian J. Exp. Biol. 27, 1094-1096). Serological methods were used effectively for diagnosis of Chrysanthemum virus B (Zaidi, A. A., Ram, R., Zaidi, S. N. H. and Mukherjee, D. (1990) Diagnosis of viruses in some ornamental plants with special reference to serological methods: New Developments. Indian Rev. Life Sci. 13, 157-174).
Enzyme linked immunosorbant assay (ELISA) and other modified form of ELISA have been extensively used for the detection of CVB from Chrysanthemum morifolium (Verma, N., Sharma, A., Ram, R., Hallan, V., Zaidi, A. A. and Garg, I. D. (2003) Detection, identification and incidence of Chrysanthemum B carlavirus in chrysanthemum in India. Crop Prot. 22, 425-429). It is highly effective in detecting the CVB from leaves. Using the DAS-ELISA, status of the viral disease was analyzed for 36 cultivars of Chrysanthemum morifolium. Some cultivars also exhibit mild leaf mottling, vein clearing or a combination of these (Hollings, M. and Stone, O. M. (1972) Chrysanthemum virus B CMI/AAB Descriptions of Plant viruses No. 110). Therefore, it is important to have reliable and quick diagnostics to diagnose the latent infection and for establishing the serological relationship between the isolates of the Chrysanthemum virus B. Similar to ELISA, Immunosorbant Electron Microscopy (ISEM) also revealed easy detection of CVB from leaves (Verma, N., Sharma, A., Ram, R., Hallan, V., Zaidi, A. A. and Garg, I. D. (2003) Detection, identification and incidence of Chrysanthemum B carlavirus in chrysanthemum in India. Crop Prot. 22, 425-429). Similar to ELISA, ISEM could detect the CVB from chrysanthemum leaves.
During last decade, RT-PCR has been used with varying degree of modification for detection of viral genome in infected plants (Yamamoto, H., Kiguchi, T. and Ohya, T. (2001) 52nd Annual Report of the Society of Plant Protection of North Japan. 85-86). Partial sequence of the Chrysanthemum virus B has been worked out and it was 3.4 kb (Levay, K. E. and Zavriev, S. K. (1991) Nucleotide sequence and gene organisation of the 3′-terminal region of Chrysanthemum virus B genomic RNA. J. Gen. Virol. 72(10), 2333-7). At IHBT, Palampur approximately 5 Kb of the CVB genome has been sequenced (EMBL Nucleotide Sequence Accession Numbers: AJ617281, AJ617282, AJ617287, AJ585240, AJ704627, AJ580956, AJ633542, AJ633540, and AJ633629). On the basis of sequencing of various geographical isolates, three biological isolates have been identified including the earlier reported Russian isolate, which resembles one of the three isolates.